Influence of specific mutations on the thermal stability of the td group I intron in vitro and on its splicing efficiency in vivo: A comparative study

ABSTRACT Group I introns are inserted into genes of a wide variety of bacteriophages of gram-positive bacteria. However, among the phages of enteric and other gram-negative proteobacteria, introns have been encountered only in phage T4 and several of its close relatives. Here we report the insertion of a self-splicing group I intron in the coding sequence of the DNA polymerase genes of ΦI and W31, phages that are closely related to T7. The introns belong to subgroup IA2 and both contain an open reading frame, inserted into structural element P6a, encoding a protein belonging to the HNH family of homing endonucleases. The introns splice efficiently in vivo and self-splice in vitro under mild conditions of ionic strength and temperature. We conclude that there is no barrier for maintenance of group I introns in phages of proteobacteria.

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Effects of glycine betaine and glycerophosphocholine on thermal stability of ribonuclease

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.4.f762 ◽

1998 ◽

Vol 274 (4) ◽

pp. F762-F765 ◽

Cited By ~ 6

Author(s):

Maurice B. Burg ◽

Eugenia M. Peters

Keyword(s):

Thermal Stability ◽

Tissue Culture ◽

Glycine Betaine ◽

Metabolic Cost ◽

Rnase A ◽

Renal Cells ◽

And Function ◽

Thermal Stability Of

Urea in renal medullas is sufficiently high to perturb macromolecules, yet the cells survive and function. The counteracting osmolytes hypothesis holds that methylamines, such as glycine betaine (betaine) and glycerophosphocholine (GPC) in renal medullas, stabilize macromolecules and oppose the effects of urea. Although betaine counteracts effects of urea on macromolecules in vitro and protects renal cells from urea in tissue culture, renal cells accumulate GPC rather than betaine in response to high urea both in vivo and in tissue culture. A proposed explanation is that GPC counteracts urea more effectively than betaine. However, we previously found GPC slightly less effective than betaine in counteracting inhibition of pyruvate kinase activity by urea. To test another macromolecule, we now compare GPC and betaine in counteracting reduction of the thermal stability of RNase A by urea. We find that urea decreases the thermal transition temperature and that betaine and GPC increase it, counteracting urea approximately equally. Therefore, the preference for GPC in response to high urea presumably has some other basis, such as a lower metabolic cost of GPC accumulation.

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Assays for the RNA chaperone activity of proteins

Biochemical Society Transactions ◽

10.1042/bst0330450 ◽

2005 ◽

Vol 33 (3) ◽

pp. 450-456 ◽

Cited By ~ 31

Author(s):

L. Rajkowitsch ◽

K. Semrad ◽

O. Mayer ◽

R. Schroeder

Keyword(s):

Group I Intron ◽

Chaperone Activity ◽

Structural Elements ◽

Rna Chaperone ◽

Group I ◽

Monomolecular Reactions ◽

Rna Chaperones ◽

Complementary Rnas

Proteins with RNA chaperone activity promote RNA folding by loosening the structure of misfolded RNAs or by preventing their formation. How these proteins achieve this activity is still unknown, the mechanism is not understood and it is unclear whether this activity is always based on the same mechanism or whether different RNA chaperones use different mechanisms. To address this question, we compare and discuss in this paper a set of assays that have been used to measure RNA chaperone activity. In some assays, this activity is related to the acceleration of monomolecular reactions such as group I intron cis-splicing or anti-termination of transcription. Hereby, it is proposed that the proteins release the RNAs from folding traps, which represent the kinetic barriers during the folding process and involve the loosening of structural elements. In most assays, however, bimolecular reactions are monitored, which include the simple acceleration of annealing of two complementary RNAs, the turnover stimulation of ribozyme cleavage and group I intron trans-splicing. The acceleration of these reactions most probably involves the unfolding of structures that interfere with annealing or folding and may in addition provoke annealing by crowding. Most assays are performed in vitro, where conditions might differ substantially from intracellular conditions, and two assays have been reported that detect RNA chaperone activity in vivo.

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Folding of the td pre-RNA with the help of the RNA chaperone StpA

Biochemical Society Transactions ◽

10.1042/bst0301175 ◽

2002 ◽

Vol 30 (6) ◽

pp. 1175-1180 ◽

Cited By ~ 11

Author(s):

O. Mayer ◽

C. Waldsich ◽

R. Grossberger ◽

R. Schroeder

Keyword(s):

Conformational Changes ◽

Three Dimensional ◽

Group I Intron ◽

Dimensional Structure ◽

Rna Chaperone ◽

Three Dimensional Structure ◽

Group I ◽

The Impact

The td group I intron is inserted in the reading frame of the thymidylate synthase gene, which is mainly devoid of structural elements. In vivo, translation of the pre-mRNA is required for efficient folding of the intron into its splicing-competent structure. The ribosome probably resolves exon-intron interactions that interfere with splicing. Uncoupling splicing from translation, by introducing a non-sense codon into the upstream exon, reduces the splicing efficiency of the mutant pre-mRNA. Alternatively to the ribosome, co-expression of genes that encode proteins with RNA chaperone activity promote folding of the td pre-mRNA in vivo. These proteins also efficiently accelerate folding of the td pre-mRNA in vitro. In order to understand the mechanism of action of RNA chaperones, we probed the impact of the RNA chaperone StpA on the structure of the td intron in vivo and compared it with that of the well characterized Cyt-18 protein, which is a group-I-intron-specific splicing factor. We found that the two proteins have opposite effects on the structure of the td intron. While StpA loosens the three-dimensional structure, Cyt-18 tightens it up. Furthermore, mutations that destabilize the intron structure render the mutants sensitive to StpA, whereas splicing of these mutants is rescued by Cyt-18. Our results provide direct evidence for protein-induced conformational changes within a catalytic RNA in vivo. Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the stabilization of the native three-dimensional structure.

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Effects of Rest and Exercise on Cardiac Blood Volume Determinations

Nuklearmedizin ◽

10.1055/s-0038-1629844 ◽

1997 ◽

Vol 36 (08) ◽

pp. 259-264

Author(s):

N. Topuzović

Keyword(s):

Radionuclide Ventriculography ◽

Left Ventricular ◽

Peak Exercise ◽

Volume Determination ◽

Group I ◽

Lv Volumes ◽

Group Ii ◽

Lv Volume

Summary Aim: The purpose of this study was to investigate the changes in blood activity during rest, exercise and recovery, and to assess its influence on left ventricular (LV) volume determination using the count-based method requiring blood sampling. Methods: Forty-four patients underwent rest-stress radionuclide ventriculography; Tc-99m-human serum albumin was used in 13 patients (Group I), red blood cells was labeled using Tc-99m in 17 patients (Group II) in vivo, and in 14 patients (Group III) by modified in vivo/in vitro method. LV volumes were determined by a count-based method using corrected count rate in blood samples obtained during rest, peak exercise and after recovery. Results: In group I at stress, the blood activity decreased by 12.6 ± 5.4%, p <0.05, as compared to the rest level, and increased by 25.1 ± 6.4%, p <0.001, and 12.8 ± 4.5%, p <0.05, above the resting level in group II and III, respectively. This had profound effects on LV volume determinations if only one rest blood aliquot was used: during exercise, the LV volumes significantly decreased by 22.1 ± 9.6%, p <0.05, in group I, whereas in groups II and III it was significantly overestimated by 32.1 ± 10.3%, p <0.001, and 10.7 ± 6.4%, p <0.05, respectively. The changes in blood activity between stress and recovery were not significantly different for any of the groups. Conclusion: The use of only a single blood sample as volume aliquot at rest in rest-stress studies leads to erroneous estimation of cardiac volumes due to significant changes in blood radioactivity during exercise and recovery.

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